Cyclic-AMP-response element binding protein (CREB) is a 43 kD nuclear transcription factor. Its transcription activity is critically dependent on phosphorylation on Ser133 to be induced by extracellular cues including growth factors and hormones. CREB is overexpressed and/or overactivated in tumor tissues of different cancer types compared to adjacent normal tissue. The goals of this application are to identify small molecule inhibitors of CREB (cyclic-AMP-response element binding protein)-mediated gene transcription and understand their mechanism of action as potential anti-cancer agents. Upon stimulation by extracellular cues, CREB is phosphorylated at Ser133 that promotes its association with CREB-binding protein (CBP) and its paralog p300 to recruit other components in the transcriptional machinery to the CREB promoter to initiate CREB-dependent gene transcription. Many oncogenic kinases including protein kinase A (PKA), mitogen-activated protein kinases (MAPKs), protein kinase B (PKB/Akt) and protein ribosomal S6 kinase (pp90RSK) can phosphorylate Ser133 in CREB. These kinases are often overactivated in cancer cells. On the other hand, three protein phosphatases, protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A) and phosphatase and tensin homolog (PTEN), can dephosphorylate CREB to attenuate CREB-mediated gene transcription. These phosphatases are often inactivated or deleted in cancer cells. Because of this dual regulation of CREB's transcription activity, CREB has been frequently observed to be overactivated in cancer cells. Furthermore, overactivation of CREB inversely correlates with cancer patients survival. Therefore, CREB is intimately implicated in tumorigenesis and has been proposed as a valuable target for developing novel cancer therapeutics. Potentially, targeting CREB can simultaneously shut down multiple oncogenic pathways, providing a novel type of cancer therapeutics with delayed or no resistance. We recently designed and synthesized a potent CREB inhibitor, 666-15, with efficacious in vitro and in vivo anti-cancer activity. In this application, we will further study 666-15 to understand its mechanism of action and structure-activity relationship (SAR). To accomplish these goals, the following three specific aims will be addressed: 1) To identify the direct molecular target(s) of 666-15; 2) To investigate the mechanisms by which Hsp60 regulates CREB's transcription; 3) To identify derivatives of 666-15 with improved physicochemical properties.